UvrD and UvrD252 counteract RecQ, RecJ, and RecFOR in a rep mutant of Escherichia coli

Rep and UvrD are two related Escherichia coli helicases, and inactivating both is lethal. Based on the observation that the synthetic lethality of rep and uvrD inactivation is suppressed in the absence of the recombination presynaptic proteins RecF, RecO, or RecR, it was proposed that UvrD is essential in the rep mutant to counteract a deleterious RecFOR-dependent RecA binding. We show here that the synthetic lethality of rep and uvrD mutations is also suppressed by recQ and recJ inactivation but not by rarA inactivation. Furthermore, it is independent of the action of UvrD in nucleotide excision repair and mismatch repair. These observations support the idea that UvrD counteracts a deleterious RecA binding to forks blocked in the rep mutant. An ATPase-deficient mutant of UvrD [uvrD(R284A)] is dominant negative in a rep mutant, but only in the presence of all RecQJFOR proteins, suggesting that the UvrD(R284A) mutant protein is deleterious when it counteracts one of these proteins. In contrast, the uvrD252 mutant (G30D), which exhibits a strongly decreased ATPase activity, is viable in a rep mutant, where it allows replication fork reversal. We conclude that the residual ATPase activity of UvrD252 prevents a negative effect on the viability of the rep mutant and allows UvrD to counteract the action of RecQ, RecJ, and RecFOR at forks blocked in the rep mutant. Models for the action of UvrD at blocked forks are proposed.     

A phcA− marker-free mutant of Ralstonia solanacearum as potential biocontrol agent of tomato bacterial wilt

A phcA⁻ mutant of Ralstonia solanacearum strain ZJ3721 was created in a marker-free method. Expression of virulence-associated genes such as xpsR, egl, tek and epsE was significantly suppressed in the phcA⁻ mutant. The ability of the mutant to control tomato bacterial wilt was evaluated by potting experiment. Results showed that application of mutant with wild type (WT) at the same time only delayed the development of wilt for about one day and the population of WT in tomato rhizosphere soil was nearly 70-fold higher than that of the mutant, resulting in a 90% disease incidence at last, as high as that of control. If the phcA⁻ mutant was applied three days earlier than WT pathogen, tomato wilt disease incidence was only 6%, 80% lower than that of control and population of WT was about 0.5-fold as much as that of mutant. Under hydroponic conditions, phcA⁻ mutant significantly triggered the expressions of genes in salicylic acid pathway but inhibited the expressions of genes in jasmonic acid (JA) and ethylene (ET) pathways. The expressions of PR-1a and GluA genes (salicylic acid pathway) in phcA⁻ mutant were 66-fold and 7.5-fold higher than in WT pathogen after three days of inoculation.     

Functional characterization of the stunt lemma palea 1 mutant allele in rice

The rice stunted lemma/palea 1 (slp1) mutant displays a dwarf, shortened panicle length, degenerated lemma and palea, and sterility. A previous study suggested that a missense mutation at the sixth amino acid of the OsSPL16 protein was likely to be responsible for the slp1 mutant phenotype. The current study shows that the overexpression of the wild-type OsSPL16 allele in slp1/slp1 and Slp1/slp1 mutants was unable to convert the slp1 mutant phenotype to normal. However, the introduction of the mutant OsSPL16 allele into a normal rice cultivar led to the slp1 mutant phenotype in transgenic plants. These results indicated that the missense mutation in OsSPL16 creates a neomorphic allele, which affects plant height and the development of the inflorescence and spikelet.     

Ecological Behavior of Lactobacillus reuteri 100-23 Is Affected by Mutation of the luxS Gene

The luxS gene of Lactobacillus reuteri 100-23C was amplified by PCR, cloned, and then sequenced. To define a physiological and ecological role for the luxS gene in L. reuteri 100-23C, a luxS mutant was constructed by insertional mutagenesis. The luxS mutant did not produce autoinducers AI-2 or AI-3. Complementation of the luxS mutation by a plasmid construct containing luxS restored AI-2 and AI-3 synthesis. In vitro experiments revealed that neither the growth rate, nor the cell yield, nor cell survival in the stationary phase were compromised in the luxS mutant relative to the wild type and complemented mutant. The ATP content of exponentially growing cells of the luxS mutant was, however, 65% of that of wild-type cells. Biofilms formed by the luxS mutant on plastic surfaces in a bioreactor were thicker than those formed by the wild type. Biofilm thickness was not restored to wild-type values by the addition of purified AI-2 to the culture medium. In vivo experiments, conducted with ex-Lactobacillus-free mice, showed that biofilms formed by the mutant strain on the epithelial surface of the forestomach were approximately twice as thick as those formed by the wild type. The ecological performance of the luxS mutant, when in competition with L. reuteri strain 100-93 in the mouse cecum, was reduced compared to that of a xylA mutant of 100-23C. These results demonstrate that LuxS influences important ecological attributes of L. reuteri 100-23C, the consequences of which are niche specific.     

Regulation of the corpora allata in the black mutant of Manduca sexta

Regulation of corpus allatum activity in the black mutant strain of Manduca sexta was studied in vivo and in vitro. Allatectomy, denervation, and implantation studies demonstrated that black mutant corpus allatum activity remains low in both wild-type and black mutant host larvae. Attempts to distinguish humoral control mechanisms versus mechanisms dependent on intact allatal nerves indicated that intact allatal nerves were not required for the reduced black mutant corpus allatum activity in vivo. Incubation of corpora allata, using [1-¹⁴C]propionate as a juvenile hormone biosynthetic precursor and haemolymph as culture medium, confirmed that black mutant corpora allata are suppressed by a factor(s) in the haemolymph. Under identical conditions wild-type corpora allata were unaffected. Finally, the lowered black mutant corpus allatum activity in haemolymph in vitro correlates with the lowered juvenile hormone titre in black mutant larvae.     

Deregulated Splicing Is a Major Mechanism of RNA-Induced Toxicity in Huntington's Disease

Huntington's disease (HD) is caused by an expanded CAG repeat in the huntingtin (HTT) gene, translating into an elongated polyglutamine stretch. In addition to the neurotoxic mutant HTT protein, the mutant CAG repeat RNA can exert toxic functions by trapping RNA-binding proteins. While few examples of proteins that aberrantly bind to mutant HTT RNA and execute abnormal function in conjunction with the CAG repeat RNA have been described, an unbiased approach to identify the interactome of mutant HTT RNA is missing. Here, we describe the analysis of proteins that preferentially bind mutant HTT RNA using a mass spectrometry approach. We show that (I) the majority of proteins captured by mutant HTT RNA belong to the spliceosome pathway, (II) expression of mutant CAG repeat RNA induces mis-splicing in a HD cell model, (III) overexpression of one of the splice factors trapped by mutant HTT ameliorates the HD phenotype in a fly model and (VI) deregulated splicing occurs in human HD brain. Our data suggest that deregulated splicing is a prominent mechanism of RNA-induced toxicity in HD.     

Use of koji prepared with a high citric acid producing mutant of Aspergillus usamii as a raw material for Saké brewing

There is a possibility of developing a new kind of saké in which the refreshing sour taste of citric acid is introduced. In this study, we bred a new mutant of Aspergillus usamii mut. shiro-usamii that produced much citric acid. The koji prepared with the mutant contained about 20 mg of citric acid per gram of dry koji, twice that of the koji of the parental strain. The activities of a-amylase, glucoamylase, and acidic protease in the koji prepared with the mutant were 82%, 94%, and 95%, respectively, those of the parental strain. Using this koji with the mutant, saké was produced. The levels of citric acid and isoamyl acetate were 5.1 and 1.4 times, respectively, those of saké prepared with koji of A. oryzae. Sensory tests indicated that saké made with koji with the mutant was refreshingly sour, with a good aroma.     

New Multiple-Deletion Method for the Corynebacterium glutamicum Genome, Using a Mutant lox Sequence

Due to the difficulty of multiple deletions using the Cre/loxP system, a simple, markerless multiple-deletion method based on a Cre/mutant lox system combining a right-element (RE) mutant lox site with a left-element (LE) mutant lox site was employed for large-scale genome rearrangements in Corynebacterium glutamicum. Eight distinct genomic regions that had been identified previously by comparative analysis of C. glutamicum R and C. glutamicum 13032 genomes were targeted for deletion. By homologous recombination, LE and RE mutant lox sites were integrated at each end of a target region. Highly efficient and accurate deletions between the two chromosomal mutant lox sites in the presence of Cre recombinase were realized. A deletion mutant lacking 190 kb of chromosomal regions, encoding a total of 188 open reading frames (ORFs), was obtained. These deletions represent the largest genomic excisions in C. glutamicum reported to date. Despite the loss of numerous predicted ORFs, the mutant exhibited normal growth under standard laboratory conditions. The Cre/loxP system using a pair of mutant lox sites provides a new, efficient genome rearrangement technique for C. glutamicum. It should facilitate the understanding of genome functions of microorganisms.     

The HtrA Protease of Campylobacter jejuni Is Required for Heat and Oxygen Tolerance and for Optimal Interaction with Human Epithelial Cells

Campylobacter jejuni is a predominant cause of food-borne bacterial gastroenteritis in the developed world. We have investigated the importance of a homologue of the periplasmic HtrA protease in C. jejuni stress tolerance. A C. jejuni htrA mutant was constructed and compared to the parental strain, and we found that growth of the mutant was severely impaired both at 44°C and in the presence of the tRNA analogue puromycin. Under both conditions, the level of misfolded protein is known to increase, and we propose that the heat-sensitive phenotype of the htrA mutant is caused by an accumulation of misfolded protein in the periplasm. Interestingly, we observed that the level of the molecular chaperones DnaK and ClpB was increased in the htrA mutant, suggesting that accumulation of nonnative proteins in the periplasm induces the expression of cytoplasmic chaperones. While lack of HtrA reduces the oxygen tolerance of C. jejuni, the htrA mutant was not sensitive to compounds that increase the formation of oxygen radicals, such as paraquat, cumene hydroperoxide, and H₂O₂. Using tissue cultures of human epithelial cells (INT407), we found that the htrA mutant adhered to and invaded human epithelial cells with a decreased frequency compared to the wild-type strain. This defect may be a consequence of the observed altered morphology of the htrA mutant. Thus, our results suggest that in C. jejuni, HtrA is important for growth during stressful conditions and has an impact on virulence.     

Morphological characteristics and gene mapping of a dense panicle (dp2) mutant in rice (Oryza sativa L.)

A dense panicle mutant (dp2) derived from the Oryza sativa ssp. japonica cultivar Nipponbare through ethyl methane sulfonate mutagenesis was used in present study. Compared to the wild type, the panicle of dp2 mutant exhibited more branches and denser grains. Further more, the number of spikelets per panicle, number of primary branches and secondary branches of dp2 mutant were significantly increased while the panicle length, and 1,000-grain weight were significantly decreased. The results from the genetic analysis indicated that the dense panicle phenotype was controlled by a single dominance nuclear gene. Polymorphic analysis of SSR and InDel markers demonstrated that the DP2 gene was located at the long arm of chromosome 2, which was further mapped between SSR markers RM341 and RM13356 in a physical region of 398 kb. Within this region, the RCN2 (LOC_Os02g32950) gene which was annotated relating to the development of rice panicle was found. Compared to the wild type, the sequence of RCN2 gene in the dp2 mutant showed that two SNPs replacement had taken place in the promoter region (G–A) and the intron region (A–T), respectively. The dp2 mutant could be a novel mutant of RCN2 gene and this novel mutant might be useful for further studies on this gene.     

Phosphoinositide-specific phospholipase C forms a complex with 14-3-3 proteins and is involved in expression of UV resistance in fission yeast

The fission yeast plc1 ⁺ gene encodes phosphoinositide-specific phospholipase C. The two- hybrid interaction assay with plexA-plc1 ⁺ as a bait revealed that Plc1p interacted with the 14-3-3 proteins Rad24p and Rad25p. Formation of a complex containing Plc1p and Rad24p in vivo was confirmed by an immunological method. As predicted from the fact that rad24 null mutant cells are hypersensitive to UV irradiation, plc1 null mutant cells were almost as sensitive to UV irradiation as rad24 null mutant cells. In addition, deletion of rad24 in the plc1 null mutant cells did not enhance the UV sensitivity, indicating that plc1 ⁺ and rad24 ⁺ belong to the same epistasis group with respect to UV sensitivity. Whereas Rad24p has been reported to be involved in the DNA damage checkpoint pathway, the delay to mitosis after UV irradiation was not defective either in rad24 null mutant cells or in plc1 null mutant cells in our analysis. Thus, Plc1p is responsible for resistance to UV irradiation, but not for the DNA damage checkpoint pathway, in cooperation with 14-3-3 proteins.     

Construction and Physiological Analysis of a Xanthomonas Mutant To Examine the Role of the oxyR Gene in Oxidant-Induced Protection against Peroxide Killing

We constructed and characterized a Xanthomonas campestris pv. phaseoli oxyR mutant. The mutant was hypersensitive to H₂O₂ and menadione killing and had reduced aerobic plating efficiency. The oxidants’ induction of the catalase and ahpC genes was also abolished in the mutant. Analysis of the adaptive responses showed that hydrogen peroxide-induced protection against hydrogen peroxide was lost, while menadione-induced protection against hydrogen peroxide was retained in the oxyR mutant. These results show that X. campestris pv. phaseoli oxyR is essential to peroxide adaptation and revealed the existence of a novel superoxide-inducible peroxide protection system that is independent of OxyR.